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The Fungal Community
Its Organization and Role
in the Ecosystem
Fourth Edition
 MYCOLOGY SERIES

Editor
J. W. Bennett
Professor
Department of Plant Biology and Pathology
Rutgers University
New Brunswick, New Jersey

Founding Editor
Paul A. Lemke

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke

2. The Fungal Community: Its Organization and Role in the Ecosystem,
edited by Donald T. Wicklow and George C. Carroll
3. Fungi Pathogenic for Humans and Animals (in three parts), edited by
Dexter H. Howard
4. Fungal Differentiation: A Contemporary Synthesis, edited by John E. Smith
5. Secondary Metabolism and Differentiation in Fungi, edited by Joan W. Bennett
and Alex Ciegler
6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy
7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin and
Michael J. Leibowitz
8. Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi,
edited by Sally A. Leong and Randy M. Berka
9. The Fungal Community: Its Organization and Role in the Ecosystem, Second
Edition, edited by George C. Carroll and Donald T. Wicklow
10. Stress Tolerance of Fungi, edited by D. H. Jennings
11. Metal Ions in Fungi, edited by Güfcnther Winkelmann and Dennis R. Winge
12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas O. Mountfort
and Colin G. Orpin
13. Fungal Genetics: Principles and Practice, edited by Cees J. Bos
14. Fungal Pathogenesis: Principles and Clinical Applications, edited by
Richard A. Calderone and Ronald L. Cihlar
15. Molecular Biology of Fungal Development, edited by Heinz D. Osiewacz
16. Pathogenic Fungi in Humans and Animals, Second Edition, edited by
Dexter H. Howard
17. Fungi in Ecosystem Processes, John Dighton
18. Genomics of Plants and Fungi, edited by Rolf A. Prade and Hans J. Bohnert
19. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural
Impacts, edited by James F. White Jr., Charles W. Bacon, Nigel L. Hywel-Jones,
and Joseph W. Spatafora
20. Handbook of Fungal Biotechnology, Second Edition, edited by Dilip K. Arora
21. Fungal Biotechnology in Agricultural, Food, and Environmental Applications,
edited by Dilip K. Arora
22. Handbook of Industrial Mycology, edited by Zhiqiang An
23. The Fungal Community: Its Organization and Role in the Ecosystem, Third Edition,
edited by John Dighton, James F. White, and Peter Oudemans
24. Fungi: Experimental Methods in Biology, Ramesh Maheshwari
25. Food Mycology: A Multifaceted Approach to Fungi and Food, edited by
Jan Dijksterhuis and Robert A. Samson
26. The Aspergilli: Genomics, Medical Aspects, Biotechnology, and Research
Methods, edited by Gustavo H. Goldman and Stephen A. Osmani
27. Defensive Mutualism in Microbial Symbiosis, edited by James F. White, Jr. and
Mónica S. Torres
28. Fungi: Experimental Methods In Biology, Second Edition, Ramesh Maheshwari
29. Fungal Cell Wall: Structure, Synthesis, and Assembly, Second Edition,
José Ruiz-Herrera
30. Polyamines in Fungi: Their Distribution, Metabolism, and Role in Cell
Differentiation and Morphogenesis, Laura Valdes-Santiago and José Ruiz-Herrera
31. Fungi in Ecosystem Processes, Second Edition, John Dighton
32. The Fungal Community: Its Organization and Role in the Ecosystem, Fourth
Edition, John Dighton and James F. White
The Fungal Community
Its Organization and Role
in the Ecosystem
Fourth Edition

edited by

John Dighton
James F. White
Front cover: Image courtesy of Björn Lindahl. Used with permission. All rights reserved.

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Library of Congress Cataloging-in-Publication Data

Names: Dighton, John, editor. | White, James F. (James Francis), 1953- ,

editor.
Title: The fungal community : its organization and role in the ecosystem /
[edited by] John Dighton and James F. White.
Description: Fourth edition. | Boca Raton : Taylor & Francis, 2016. | Series:
Mycology series
Identifiers: LCCN 2016027059| ISBN 9781498706650 (hardback : alk. paper) |
ISBN a 9781498706674 (e-book)
Subjects: LCSH: Fungal communities. | Fungi-- Ecology.
Classification: LCC QK604.2.C64 F86 2016 | DDC 579.5-- dc23
LC record available at https://lccn.loc.gov/2016027059

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 Contents
Introduction ...........................................................................................................................................................................xiii
Editors .................................................................................................................................................................................... xv
Contributors ......................................................................................................................................................................... xvii

Part I
Integrating Genomics and Metagenomics into Community Analysis

Chapter 1
Molecular Community Ecology of Arbuscular Mycorrhizal Fungi ........................................................................................ 3
Joe D. Taylor, Thorunn Helgason, and Maarja Öpik

Chapter 2
Comparative and Functional Genomics of Ectomycorrhizal Symbiosis ............................................................................... 27
Joske Ruytinx and Francis Martin

Chapter 3
Early Fungi: Evidence from the Fossil Record ...................................................................................................................... 37
Michael Krings, Thomas N. Taylor, and Carla J. Harper

Chapter 4
Evolution of Lichens ...............................................................................................................................................................53
H. Thorsten Lumbsch and Jouko Rikkinen

Part II
Recent Advances in Fungal Endophyte Research

Chapter 5
A Novel Framework for Decoding Fungal Endophyte Diversity .......................................................................................... 65
Natalie Christian, Briana K. Whitaker, and Keith Clay

Chapter 6
Foliar Endophyte Communities and Leaf Traits in Tropical Trees ....................................................................................... 79
Sunshine Van Bael, Catalina Estrada, and A. Elizabeth Arnold

Chapter 7
Community Assembly of Phyllosphere Endophytes: A Closer Look at Fungal Life Cycle Dynamics, Competition,
and Phytochemistry in the Shaping of the Fungal Community ............................................................................................ 95
Christopher B. Zambell and James F. White

Chapter 8
Interactions between Fungal Endophytes and Bacterial Colonizers of Fescue Grass ......................................................... 109
Elizabeth Lewis Roberts and Christopher Mark Adamchek

vii
viii CONTENTS

Part III
Fungal Communities in Terrestrial Ecosystems

Chapter 9
Geomycology: Geoactive Fungal Roles in the Biosphere.....................................................................................................121
Geoffrey Michael Gadd

Chapter 10
Lichens and Microfungi in Biological Soil Crusts: Structure and Function Now and in the Future ...................................137
Jayne Belnap and Otto L. Lange

Chapter 11
Ecology of Fungal Phylloplane Epiphytes ............................................................................................................................159
Katalin Malcolm and John Dighton

Chapter 12
Wood Decay Communities in Angiosperm Wood ...............................................................................................................169
Lynne Boddy, Jennifer Hiscox, Emma C. Gilmartin, Sarah R. Johnston, and Jacob Heilmann-Clausen

Chapter 13
Lichens in Natural Ecosystems .............................................................................................................................................191
Darwyn Coxson and Natalie Howe

Part IV
Fungal Communities in Marine and Aquatic Ecosystems

Chapter 14
Diversity and Role of Fungi in the Marine Ecosystem ........................................................................................................ 207
Chandralata Raghukumar

Chapter 15
Aquatic Hyphomycete Communities in Freshwater ............................................................................................................ 225
Kandikere R. Sridhar

Chapter 16
The Ecology of Chytrid and Aphelid Parasites of Phytoplankton....................................................................................... 239
Thomas G. Jephcott, Floris F. van Ogtrop, Frank H. Gleason, Deborah J. Macarthur, and Bettina Scholz

Chapter 17
Crown Oomycetes Have Evolved as Effective Plant and Animal Parasites ........................................................................ 257
Agostina V. Marano, Frank H. Gleason, Sarah C. O. Rocha, Carmen L. A. Pires-Zottarelli, and José I. de Souza
CONTENTS ix

Part V
Fungal Adaptations to Stress and Conservation

Chapter 18
Adaptations of Fungi and Fungal-Like Organisms for Growth under Reduced Dissolved Oxygen Concentrations ...........275
Sandra Kittelmann, Cathrine S. Manohar, Ray Kearney, Donald O. Natvig, and Frank H. Gleason

Chapter 19
Fungi in Extreme and Stressful Environments .................................................................................................................... 293
Sharon A. Cantrell

Chapter 20
Reaching the Wind: Boundary Layer Escape as a Constraint on Ascomycete Spore Dispersal......................................... 309
Anne Pringle, Michael Brenner, Joerg Fritz, Marcus Roper, and Agnese Seminara

Chapter 21
Who Cares? The Human Perspective on Fungal Conservation ............................................................................................321
Elizabeth S. Barron

Part VI
Fungal–Faunal Interactions

Chapter 22
Belowground Trophic Interactions........................................................................................................................................333
Amy Treonis

Chapter 23
Mycophagy and Spore Dispersal by Vertebrates ................................................................................................................. 347
Alessandra Zambonelli, Francesca Ori, and Ian Hall

Chapter 24
The Fungal Spore: Myrmecophilous Ophiocordyceps as a Case Study ...............................................................................359
João P. M. Araújo and David P. Hughes

Chapter 25
Coevolution of Fungi and Invertebrates ............................................................................................................................... 369
Xingzhong Liu, Lin Wang, and Meichun Xiang

Chapter 26
Fungal Diversity of Macrotermes–Termitomyces Nests in Tsavo, Kenya ........................................................................... 377
Jouko Rikkinen and Risto Vesala
x CONTENTS

Chapter 27
Emerging Mycoses and Fungus-Like Diseases of Vertebrate Wildlife ............................................................................... 385
Hannah T. Reynolds, Daniel Raudabaugh, Osu Lilje, Matthew Allender, Andrew N. Miller,
and Frank H. Gleason

Chapter 28
Geomyces and Pseudogymnoascus: Emergence of a Primary Pathogen, the Causative Agent of Bat White-Nose
Syndrome ............................................................................................................................................................................. 405
Michelle L. Verant, Andrew M. Minnis, Daniel L. Lindner, and David S. Blehert

Part VII
Fungal Communities, Climate Change, and Pollution

Chapter 29
Mycorrhizal Fungi and Accompanying Microorganisms in Improving Phytoremediation Techniques ..............................419
Piotr Rozpądek, Agnieszka Domka, and Katarzyna Turnau

Chapter 30
Effects of Toxic Metals on Chytrids, Fungal-Like Organisms, and Higher Fungi ..............................................................433
Linda Henderson, Erna Lilje, Katie Robinson, Frank H. Gleason, and Osu Lilje

Chapter 31
The Fungal Community in Organically Polluted Systems ...................................................................................................459
Hauke Harms, Lukas Y. Wick, and Dietmar Schlosser

Chapter 32
Fungal Communities and Climate Change ...........................................................................................................................471
Jennifer M. Talbot

Part VIII
Fungi in the Built Environment

Chapter 33
Decomposition of Wooden Structures by Fungi ...................................................................................................................491
Benjamin W. Held

Chapter 34
Fungal Degradation of Our Cultural Heritage ......................................................................................................................501
John Dighton

Chapter 35
Microorganisms for Safeguarding Cultural Heritage .......................................................................................................... 509
Edith Joseph, Saskia Bindschedler, Monica Albini, Lucrezia Comensoli, Wafa Kooli, and Lidia Mathys
CONTENTS xi

Part IX
Fungal Signaling and Communication

Chapter 36
Airborne Signals: Volatile-Mediated Communication between Plants, Fungi, and Microorganisms .................................521
Samantha Lee, Guohua Yin, and Joan W. Bennett

Chapter 37
Mycorrhizal Fungal Networks as Plant Communication Systems .......................................................................................539
David Johnson and Lucy Gilbert

Chapter 38
Fungal–Fungal Interactions: From Natural Ecosystems to Managed Plant Production, with Emphasis on Biological
Control of Plant Diseases ..................................................................................................................................................... 549
Dan Funck Jensen, Magnus Karlsson, and Björn D. Lindahl

Chapter 39
Ecology and Evolution of Fungal-Bacterial Interactions ..................................................................................................... 563
Stefan Olsson, Paola Bonfante, and Teresa E. Pawlowska
Index .................................................................................................................................................................................... 585

In all subjects in science, new findings and use of new tech-
nologies allow us to develop an ever-greater understanding of
our world. With the evolution of molecular tools for identify-
ing fungi and genomics to understand relationships between
fungal species, the entire concept of fungal taxonomy has
been changed from classical Linnean nomenclature to that of
bar codes and robust multigene phylogenetic molecular trees
(Hibbett and Taylor 2013; Money 2013). These tools allow
us to ask new questions in relation to the evolution of the
kingdom Fungi and the major taxa within. Along with this
surge, the development of molecular tools in the application
of genomics, metagenomics, and transcriptomics has allowed
us to understand more about the functional aspects of fungi in
real time (Martin 2014).
Fungi interact with all components of the ecosystems on
the earth, but do not act alone. Different fungi interact with
each other and with other organisms in both trophic and non-
trophic interactions. They also interact with abiotic compo-
nents of ecosystems and with pollutants that humans produce
(Dighton 2016). Indeed, interaction of fungi with humans
has led to a whole new area of study of the “built environ-
ment” as an ecosystem where fungi are under stress and have
evolved to survive (Gostincar et al. 2011) and cause concern
over human health and the integrity of their belongings.
In this edition of The Fungal Community, we have
attempted to compile several sections that each represent
some recent advances in thinking.
The section “Integrating Genomics and Metagenomics
into Community Analysis” explores the use of molecular
techniques to characterize fungal communities and some of
the underlying genetic regulation of processes establishing
symbioses. Some of these methods are then explored in rela-
tion to the evolution of fungi and fungal groups.
The study of endophytic microbes continues to be popu-
lar, due to their presumed important, but mysterious, roles
as part of plant microbiomes—and their potential to impact
agricultural crops as agents of plant growth promotion,
and biotic and abiotic defense. Recent advances in fungal
endophyte research explore what little is currently under-
stood regarding the biologies and ecologies of endophytic
microbes, how they interact with host cells and tissues, and
how they are regulated within plants. This section focuses
on the symbiosis between fungal endophytes and plants to
explore fungal endophyte diversity, classification, and how
endophytes interact with one another and plant hosts.
The role of fungal communities in natural ecosystems
includes terrestrial, aquatic, and marine ecosystems and is
explored here in two sections (terrestrial and aquatic and
marine). As stated earlier, fungi are involved in many func-
tions in the natural ecosystem including interactions with
both the abiotic and biotic components. Fungi are important
players in terrestrial, freshwater, and marine ecosystems
Introduction
where they are involved in soil development, soil fertility by
decomposing plant and animal remains and cycling nutrients,
and as pathogens of both plants and animals. In extreme envi-
ronments, fungi have developed physiological attributes to
allow them to exist in cold, saline, and low-oxygen conditions.
Some of these attributes are discussed in the section “Fungal
Adaptations to Stress and Conservation.” Fungal fruiting and
sporulation may be associated with stress; fungi are able to
disseminate spores through unique structural adaptations of
the fruiting bodies. Ecosystem functions require a diverse
assemblage of fungi, and there is concern that human influ-
ences on land use management (agricultural and forests) may
make many fungal species vulnerable to extinction. Compared
to more charismatic plants and animals, the question can be
asked “Who cares?” when it comes to fungal conservation.
Interactions between fungi and animals may be trophic
or parasitic in nature. In the same way that fungi can form a
significant part of the human diet, this can become even more
pronounced in both invertebrate and vertebrate fauna. Not only
do fungi provide nutrients to animals consuming them, the
animals may also serve as vectors of fungal spores allowing
for dissemination. The interactions between animals and their
pathogenic fungi may be closely tied in evolutionary terms.
With the increased movement of organisms around the world,
exotic fungi have become more potent pathogens of their ani-
mal hosts and a range of emerging fungal diseases of animals
is explored in the “Fungal–Faunal Interactions” section.
Human influences on the environment have been dra-
matic. Through industrial processes, the construction of
urban habitats and extensive transportation systems, we have
polluted the environment with toxic wastes of heavy metals,
radionuclides, and organic chemicals. Through vehicular
traffic, we have increased the availability of inorganic nitro-
gen in excess of the ecosystem stoichiometric balance and
altered the global carbon cycle. The chapters in the section
“Fungal Communities, Climate Change, and Pollution” dis-
cuss metal toxicity in the terrestrial and aquatic ecosystems,
the influence of fungi in organically polluted ecosystems,
and the influence of climate change on fungi.
Continuing the theme of human impacts, “Fungi in the Built
Environment” looks at aspects of fungal communities in the
built environment. These subjects range from the destruction
of buildings through the potential loss of cultural heritage by
fungal spoilage of artifacts to novel methods of bio-protection
of artifacts. Much more could be said on this subject with
respect to health issues caused by fungi growing in our build-
ings, but this is considered outside the scope of this volume.
Autecology is the study of the individual species in the
ecosystem. These studies are, however, somewhat unre-
alistic given that any one organism interacts with multiple
other organisms in the ecosystem; thus, a synecological
approach is necessary to understand these interactions and
xiii
xiv
their functional consequences. In the final section, “Fungal
Signaling and Communication,” we explore a number of
interactions with an emphasis on the way in which com-
munication between organisms is an essential part of these
interactions.
Our increasing understanding of genetic regulation of
protein expression has led to a number of new advances
in the elucidation of pathways of communication between
fungi and between fungi and other organisms.
It is impossible to delve into all aspects of fungal
community interactions and functions in one volume. We
hope that these chapters will provide a framework for
understanding some of these areas of research and that
the outlooks from these chapters will stimulate further
research.
During the production of this book we were informed
of the death of one of our authors, Thomas N. Taylor, who
INTRODUCTION
will be greatly missed in the mycological community. Our
sympathies go out to his family
REFERENCES
Dighton, J. 2016. Fungi in Ecosystem Processes, 2nd Edition, Boca
Raton, FL, CRC Press/Taylor & Francis.
Gostincar, C., M. Grube, and N. Gunde-Cimerman. 2011. Evolution
of fungal pathogens in domestic environments? Fungal
Biology 115:1008–1018.
Hibbett, D. S. and J. W. Taylor. 2013. Fungal systematics: Is a new
age of enlightenment at hand? Nature Reviews Microbiology.
doi:10.1038/nrmicro2942.
Martin, F. (Ed.). 2014. The Ecological Genomics of Fungi. Oxford,
UK, John Wiley & Sons.
Money, N. P. 2013. Against naming fungi. Fungal Biology
117:163–465.

John Dighton, PhD, earned an MSc degree in ecology
at Durham University (Durham, United Kingdom), and a
PhD from London University. After a brief spell of teach-
ing high school, he worked for 15 years for the Institute of
Terrestrial Ecology, Merlewood (United Kingdom) (Natural
Environment Research Council), where he worked on ecto-
mycorrhizal fungi, forest soil ecology, forest nutrition, and
impacts of pollutants on fungi. He moved to the United States
to work at Rutgers University (New Brunswick, New Jersey),
where he holds a spilt appointment between the Department
of Ecology, Evolution and Natural Resources (SEBS) and
the Biology Department in Camden. Dr. Dighton is also the
director of the Rutgers Pinelands Field Station in the New
Jersey pine barrens. Here, he has continued his research
in forest ecology and mycology and interactions with pol-
lutants and disturbance. He teaches courses in mycology
and soil ecology at two campuses of the university. He has
published more than 100 scientific papers and has served
Editors
on the editorial boards of Soil Biology and Biochemistry,
Fungal Biology, Fungal Ecology, and Bartonia. Dr. Dighton
has written and edited books on soil ecology and mycology-
related topics.
James F. White, PhD, is a professor in the Department
of Plant Biology and Pathology at Rutgers University. He
obtained BS and MS degrees in botany and plant pathology,
respectively, from Auburn University (Alabama) and received
a PhD degree in botany from the University of Texas (Austin).
Dr. White has published more than 200 research articles
and book chapters on the topic of endophytic microbes
and has edited 5 books on that and related topics. He also
teaches graduate and undergraduate courses in mycology.
Dr. White’s research accomplishment has been acknowledged
by his election as a Fellow of the American Association for
the Advancement of Science and receipt of the Alexopoulos
Prize from the Mycological Society of America.
xv
 Contributors
Christopher Mark Adamchek Lynne Boddy
Department of Biology School of Biosciences
Southern Connecticut State University Cardiff University
New Haven, Connecticut Cardiff, United Kingdom

Monica Albini Paola Bonfante

Laboratory of Microbiology Department of Life Sciences & Systems Biology
University of Neuchâtel University of Torino
Neuchâtel, Switzerland Torino, Italy

Matthew Allender Michael Brenner

Wildlife Epidemiology Laboratory School of Engineering and Applied Sciences
Department of Veterinary Clinical Medicine Harvard University
College of Veterinary Medicine Cambridge, Massachusetts
University of Illinois
Urbana, Illinois Sharon A. Cantrell
Department of Biology
João P. M. Araújo School of Natural Science and Technology
Department of Biology Universidad del Turabo
Penn State University Gurabo, Puerto Rico
University Park, Pennsylvania
Natalie Christian
A. Elizabeth Arnold Evolution, Ecology and Behavior Program
School of Plant Sciences Department of Biology
Department of Ecology and Evolutionary Biology Indiana University
The University of Arizona Bloomington, Indiana
Tucson, Arizona
Keith Clay
Elizabeth S. Barron Evolution, Ecology and Behavior Program
Department of Geography and Urban Planning Department of Biology
University of Wisconsin Oshkosh Indiana University
Oshkosh, Wisconsin Bloomington, Indiana

Jayne Belnap Lucrezia Comensoli

U.S. Geological Survey Laboratory of Microbiology
Moab, Utah University of Neuchâtel
Neuchâtel, Switzerland
Joan W. Bennett
Department of Plant Biology Darwyn Coxson
Rutgers University Ecosystem Science and Management Program
New Brunswick, New Jersey University of Northern British Columbia
British Columbia, Canada
Saskia Bindschedler
Laboratory of Microbiology José I. de Souza
University of Neuchâtel Instituto de Botânica
Neuchâtel, Switzerland Núcleo de Pesquisa em Micologia
São Paulo, Brazil
David S. Blehert
USGS—National Wildlife Health Center John Dighton
Madison, Wisconsin Rutgers University Pinelands Field Station
New Lisbon, New Jersey

xvii
xviii CONTRIBUTORS

Agnieszka Domka Carla J. Harper

Institute of Environmental Sciences Department für Geo- und Umweltwissenschaften,
Jagiellonian University Paläontologie und Geobiologie
Kraków, Poland Ludwig-Maximilians-Universität München
and
Catalina Estrada Bayerische Staatssammlung für Paläontologie und
Department of Life Sciences Geologie
Imperial College London Munich, Germany
Silwood Park, United Kingdom and
Department of Ecology and Evolutionary Biology
Joerg Fritz Natural History Museum and Biodiversity Institute
School of Engineering and Applied Sciences University of Kansas
Harvard University Lawrence, Kansas
Cambridge, Massachusetts
Jacob Heilmann-Clausen
Geoffrey Michael Gadd Center for Macroecology, Evolution and Climate
Geomicrobiology Group Natural History Museum of Denmark
School of Life Sciences University of Copenhagen
University of Dundee Copenhagen, Denmark
Dundee, United Kingdom
Benjamin W. Held
Lucy Gilbert Department of Plant Pathology
Ecological Sciences Group University of Minnesota
James Hutton Institute St. Paul, Minnesota
Aberdeen, United Kingdom
Thorunn Helgason
Department of Biology
Emma C. Gilmartin University of York
Cardiff School of Biosciences York, United Kingdom
Cardiff University
Cardiff, United Kingdom Linda Henderson
School of Life and Environmental Sciences
Frank H. Gleason University of Sydney
School of Biological Sciences Sydney, Australia
University of Sydney
Sydney, Australia Jennifer Hiscox
Cardiff School of Biosciences
Ian Hall Cardiff University
Truffles and Mushrooms (Consulting) Ltd. Cardiff, United Kingdom
Dunedin, New Zealand
Natalie Howe
Department of Ecology, Evolution and Natural Resources
Hauke Harms Rutgers University
Department of Environmental Microbiology New Brunswick, New Jersey
Helmholtz Centre for Environmental Research
Leipzig, Germany David P. Hughes
Department of Biology
and
Department of Entomology
Penn State University
University Park, Pennsylvania
CONTRIBUTORS xix

Dan Funck Jensen Michael Krings

Department of Forest Mycology and Plant Pathology Department für Geo- und Umweltwissenschaften,
Uppsala BioCenter Paläontologie und Geobiologie
Swedish University of Agricultural Sciences Ludwig-Maximilians-Universität München
Uppsala, Sweden and
Bayerische Staatssammlung für Paläontologie und
Thomas G. Jephcott Geologie
Faculty of Agriculture and Environment Munich, Germany
Department of Environmental Sciences
and
University of Sydney
Sydney, Australia Department of Ecology and Evolutionary Biology
Natural History Museum and Biodiversity Institute
David Johnson University of Kansas
Institute of Biological and Environmental Sciences Lawrence, Kansas
University of Aberdeen
Aberdeen, United Kingdom Otto L. Lange
Department of Biology
Sarah R. Johnston University of Würzburg
Cardiff School of Biosciences Würzburg, Germany
Cardiff University
Cardiff, United Kingdom Samantha Lee
Department of Plant Biology
Edith Joseph Rutgers University
Laboratory of Microbiology New Brunswick, New Jersey
University of Neuchâtel
and Erna Lilje
Haute Ecole Arc Conservation-Restauration School of Life and Environmental Sciences
Neuchâtel, Switzerland University of Sydney
Sydney, Australia
Magnus Karlsson
Department of Forest Mycology and Plant Pathology Osu Lilje
Uppsala BioCenter School of Life and Environmental Sciences
Swedish University of Agricultural Sciences University of Sydney
Uppsala, Sweden Sydney, Australia

Ray Kearney Björn D. Lindahl

Department of Infectious Diseases and Immunology Department of Soil and Environment
The University of Sydney Swedish University of Agricultural Sciences
Sydney, Australia Uppsala, Sweden

Sandra Kittelmann Daniel L. Lindner

AgResearch Ltd. U.S. Forest Service
Grasslands Research Centre Center for Forest Mycology Research
Palmerston North, New Zealand Madison, Wisconsin

Wafa Kooli Xingzhong Liu

Laboratory of Microbiology Institute of Microbiology
University of Neuchâtel State Key Laboratory of Mycology
Neuchâtel, Switzerland Chinese Academy of Sciences
Beijing, China

H. Thorsten Lumbsch
Integrative Research Center, Science & Education
The Field Museum
Chicago, Illinois
xx CONTRIBUTORS

Deborah J. Macarthur Maarja Öpik

School of Science Institute of Ecology and Earth Sciences
Faculty of Health Sciences University of Tartu
Australian Catholic University Tartu, Estonia
Sydney, Australia
Francesca Ori
Katalin Malcolm Department of Life, Health and Environmental Sciences
Department of Ecology, Evolution and Natural Resources University of L’Aquila
Rutgers University L’Aquila, Italy
New Brunswick, New Jersey
Teresa E. Pawlowska
Cathrine S. Manohar School of Integrative Plant Science, Plant Pathology &
Biological Oceanography Division Plant-Microbe Biology
Council of Scientific and Industrial Research—National Cornell University
Institute of Oceanography Ithaca, New York
Goa, India
Carmen L. A. Pires-Zottarelli
Agostina V. Marano Instituto de Botânica
Instituto de Botânica Núcleo de Pesquisa em Micologia
Núcleo de Pesquisa em Micologia São Paulo, Brazil
São Paulo, Brazil
Anne Pringle
Francis Martin Departments of Botany and Bacteriology
Institut National de la Recherche Agronomique University of Wisconsin–Madison
Université de Lorraine Interactions Arbres/ Madison, Wisconsin
Microorganismes
Laboratoire d’Excellence ARBRE Chandralata Raghukumar
Champenoux, France National Institute of Oceanography
Goa, India
Lidia Mathys
Laboratory of Microbiology Daniel Raudabaugh
University of Neuchâtel Department of Plant Biology
Neuchâtel, Switzerland University of Illinois
Urbana, Illinois
Andrew N. Miller
and
Illinois Natural History Survey
University of Illinois Illinois Natural History Survey
Champaign, Illinois University of Illinois
Champaign, Illinois
Andrew M. Minnis
U.S. Forest Service Hannah T. Reynolds
Center for Forest Mycology Research Department of Plant Pathology
Madison, Wisconsin The Ohio State University
Columbus, Ohio
Donald O. Natvig
Department of Biology Jouko Rikkinen
University of New Mexico Department of Biosciences
Albuquerque, New Mexico University of Helsinki
Helsinki, Finland
Stefan Olsson
State Key Laboratory of Ecological Pest Control Elizabeth Lewis Roberts
for Fujian and Taiwan Crops Department of Biology
Fujian Agriculture and Forestry University Southern Connecticut State University
Fuzhou, China New Haven, Connecticut
CONTRIBUTORS xxi

Katie Robinson Agnese Seminara

School of Life and Environmental Sciences Laboratoire de Physique de la Matière Condensée
University of Sydney Université Côte d’Azur and CNRS
Sydney, Australia Nice, France

Sarah C. O. Rocha Kandikere R. Sridhar

Instituto de Botânica Department of Biosciences
Núcleo de Pesquisa em Micologia Mangalore University, Mangalagangotri
São Paulo, Brazil Mangalore, India

Marcus Roper Jennifer M. Talbot

Department of Mathematics Deptment of Biology
University of California Boston University
Los Angeles, California Boston, Massachusetts

Joe D. Taylor
Piotr Rozpądek Department of Biology
Institute of Environmental Sciences University of York
Jagiellonian University York, United Kingdom
Kraków, Poland
and Thomas N. Taylor (Deceased)
Department of Ecology and Evolutionary Biology
Institute of Plant Physiology Natural History Museum and Biodiversity Institute
Polish Academy of Sciences University of Kansas
Kraków, Poland Lawrence, Kansas

Joske Ruytinx Amy Treonis

Institut National de la Recherche Agronomique Department of Biology
Université de Lorraine Interactions Arbres/ University of Richmond
Microorganismes Richmond, Virginia
Laboratoire d’Excellence ARBRE
Champenoux, France Katarzyna Turnau
Institute of Environmental Sciences
and
and
Centrum voor Milieukunde, Milieubiologie Malopolska Centre of Biotechnology
Universiteit Hasselt Jagiellonian University
Centrum voor Milieukunde, Milieubiologie Kraków, Poland
Diepenbeek, Belgium
Sunshine Van Bael
Dietmar Schlosser Department of Ecology and Evolutionary Biology
Department of Environmental Microbiology Tulane University
Helmholtz Centre for Environmental Research New Orleans, Louisiana
Leipzig, Germany and
Smithsonian Tropical Research Institute
Bettina Scholz Apartado, Republic of Panama
BioPol ehf
Skagaströnd, Iceland Floris F. van Ogtrop
and Department of Environmental Sciences
Faculty of Agriculture and Environment
Faculty of Natural Resource Sciences University of Sydney
University of Akureyri Sydney, Australia
Akureyri, Iceland
xxii
Michelle L. Verant
Department of Pathobiological Sciences
School of Veterinary Medicine
University of Wisconsin–Madison
Madison, Wisconsin
and
National Park Service
Biological Resources Division
Wildlife Health
Fort Collins, Colorado
Risto Vesala
Department of Biosciences
University of Helsinki
Helsinki, Finland
Lin Wang
State Key Laboratory of Mycology
Institute of Microbiology
Chinese Academy of Sciences
Beijing, China
Briana K. Whitaker
Evolution, Ecology and Behavior Program
Department of Biology
Indiana University
Bloomington, Indiana
CONTRIBUTORS
James F. White
Department of Plant Biology & Pathology
Rutgers University
New Brunswick, New Jersey
Lukas Y. Wick
Department of Environmental Microbiology
Helmholtz Centre for Environmental Research
Leipzig, Germany
Meichun Xiang
State Key Laboratory of Mycology
Institute of Microbiology
Chinese Academy of Sciences
Beijing, China
Guohua Yin
Department of Plant Biology & Pathology
Rutgers University
New Brunswick, New Jersey
Christopher B. Zambell
Department of Plant Biology & Pathology
Rutgers University
New Brunswick, New Jersey
Alessandra Zambonelli
Dipartimento di Scienze Agrarie
University of Bologna
Bologna, Italy
 PART I

Integrating Genomics and Metagenomics

into Community Analysis
 CHAPTER 1

Molecular Community Ecology of

Arbuscular Mycorrhizal Fungi

Joe D. Taylor, Thorunn Helgason, and Maarja Öpik

CONTENTS

1.1 Introduction .................................................................................................................................................................... 3

1.2 Contribution of Molecular Methods to Understanding AM Fungal Diversity ............................................................... 4
1.2.1 From Morphology to Molecules ......................................................................................................................... 4
1.2.2 From Community Fingerprinting to Deep Sequencing ...................................................................................... 4
1.2.3 From Taxonomic Expert Knowledge to Sequence Databases ............................................................................ 6
1.2.4 Molecular Quantification of AM Fungi.............................................................................................................. 6
1.2.5 Stable Isotope Probing for AM Research ........................................................................................................... 7
1.2.6 Analysis of Physiologically Active AM Fungal Communities ........................................................................... 8
1.3 Sampling AM Fungi to Study Taxonomic and Functional Diversity ............................................................................. 9
1.3.1 Sampling Design................................................................................................................................................. 9
1.3.2 Sample Preservation ..........................................................................................................................................10
1.3.3 DNA and RNA Extraction from AM Fungal Samples ......................................................................................11
1.4 High-Throughput Sequencing for AM Fungal Research.............................................................................................. 12
1.4.1 Marker Choice .................................................................................................................................................. 12
1.4.2 HTS Platform Choice ....................................................................................................................................... 13
1.4.3 Bioinformatics and Databases ...........................................................................................................................14
1.5 Taxonomy, Phylogeny, and Genomics of AM Fungi .................................................................................................... 15
1.5.1 Historical and Current Taxonomy .................................................................................................................... 15
1.5.2 Genomic and Multigene Data ............................................................................................................................16
1.6 Metagenomics and Metatranscriptomics for AM Fungal Research ..............................................................................16
1.6.1 Metagenomics ....................................................................................................................................................16
1.6.2 Metatranscriptomics ..........................................................................................................................................17
1.7 Outlook ..........................................................................................................................................................................17
References ...............................................................................................................................................................................18

1.1 INTRODUCTION
The arbuscular mycorrhiza (AM) is a symbiosis between
fungi of the phylum Glomeromycota and roots or belowground
organs of plants (Smith and Read 2008). Approximately, two-
thirds of plant species form AM symbiosis (Wang and Qiu
2006; Smith and Read 2008). Arbuscular mycorrhizal fungi are
obligate symbionts and rely on carbon sources obtained from the
photosynthetic partner (Fitter et al. 1998). Host plants receive
phosphorus (Javot et al. 2007; Smith et al. 2015a) and nitrogen
(Hodge and Storer 2015) via AM fungal partners and frequently
show positive growth responses to AM fungi (Artursson et al.
2006). In addition, AM plants can show increased resistance
to biotic stress, such as pathogens (Jung et al. 2012; Pozo et al.
2015) and herbivores (Vannette and Rasmann 2012), and
abiotic stress, such as salinity, drought, and increased heavy
metal concentrations (Smith and Read 2008; Porcel et al.
2012). At the community and ecosystem levels, AM fungal
diversity is positively related to plant diversity and productiv-
ity (van der Heijden et al. 1998, 2008; Hiiesalu et al. 2014).

3
4 THE FUNGAL COMMUNITY
The benefits that host plants gain from the AM interaction
depend on the identities of both plants and AM fungi involved.
There is evidence that AM fungal species and isolates can
differ in terms of benefits provided to the host (Munkvold
et al. 2004). Arbuscular mycorrhizal fungi potentially have
a large impact on the competitive interactions between plant
species (Facelli et al. 2010; Moora and Zobel 2010). However,
meta-analysis of various studies has shown that analysis of
such benefits is incredibly complex and involves a multitude
of biotic and abiotic factors (Hoeksema et al. 2010). Thus, it
has been proposed that diversity of AM fungal communities
may be a major driver of the dynamics of terrestrial plant
communities (van der Heijden and Cornelissen 2002; Bever
et al. 2010; Klironomos et al. 2011; Zobel and Öpik 2014).
Arbuscular mycorrhizal fungal communities are now
being studied both to gather empirical information about
their patterns of composition and abundance and to under-
stand the underlying factors generating the patterns (Öpik
et al. 2006, 2010; Dumbrell et al. 2010; Kivlin et al. 2011;
Davison et al. 2015). Our knowledge of AM fungal field
ecology has increased markedly in the past two decades with
the development of molecular biology techniques and their
increasing accessibility to mycorrhizal ecologists. Due to the
microscopic size, shortage of morphological traits suitable
for identification of field material, and limited knowledge
about the natural history of AM fungi, studying their large-
scale dynamics on the basis of micromorphological methods
has made slow progress. Molecular techniques revolution-
ized the AM fungal community ecology research by making
rapid community-level analysis possible. Further develop-
ment of molecular techniques and molecular data analysis
approaches, specifically high-throughput sequencing (HTS),
is allowing field-based community ecology of AM fungi
increasingly more feasible, reliable, and reproducible.
This chapter summarizes the analysis of AM fun-
gal communities by using modern molecular techniques,
describing where molecular techniques have provided new
knowledge and enabled major discoveries, providing a short
guide to functional analysis of AM fungal communities by
using molecular techniques, and presenting an outlook for
the future. In particular, we aim to highlight how molecu-
lar techniques can move the field of AM fungal community
ecology from focusing on taxonomic diversity to function-
ing and enabling research questions such as “Who is there?”
to be followed by “ … and what are they doing?”
1.2 CONTRIBUTION OF MOLECULAR METHODS
TO UNDERSTANDING AM FUNGAL DIVERSITY
1.2.1 From Morphology to Molecules
Assessment of AM fungal diversity and dynamics has
been one of the major foci of research into the ecology of
AM fungi. Microscopy-based studies paved the way for AM
fungal research, raising many questions that molecular tech-
niques have since been able to answer. Early work was based
solely on microscopical identification of AM fungal spores
sampled from field-collected soil or multiplied in trap cultures
(Mosse 1973). This is a slow process, relying heavily on expert
knowledge. Furthermore, the spore-based detection of AM
fungi has its known limitations (Sanders 2004). Importantly,
spores of AM fungi are resting and dispersal organs, and fac-
tors driving sporulation are not well understood. Thus, the
presence of AM fungal spores is evidence of the species pres-
ent, but the absence of spores is not evidence of the absence
of species (e.g., Clapp et al. 1995; Varela-Cervero et al. 2015).
Instead, this indicates the absence of sporulation.
The importance of spore-based studies to AM fungal
community ecology for gathering observational evidence
and developing and answering essential questions cannot be
underestimated. Such studies showed that AM fungal diver-
sity can have seasonal and spatial patterns (Merryweather
and Fitter 1998; Zangaro et al. 2013), including successional
dynamics (Johnson et al. 1991). The first field evidence to
which AM fungal diversity and plant diversity were related
was based on soil spore identification (Landis et al. 2004).
Sporulation dynamics provided data for developing the con-
cept of plant-AM fungal feedback (Bever 1994; Bever et al.
1997) and differential host-AM fungal relationships (Bever
et al. 1996). Not only have spore-based studies provided us
with important field-based observations, but sporulating and
culturable AM fungi are an important source of clean mate-
rial for conducting experiments in controlled conditions
(van der Heijden et al. 1998) and for genomics, genetics, and
physiology of AM fungi (e.g., Tisserant et al. 2012, 2013;
Lin et al. 2014).
Molecular techniques are currently the prevailing
approach for studying AM fungal communities. Compared
to studying AM fungal spores, the deoxyribonucleic acid
(DNA)- and particularly ribonucleic acid (RNA)-based
methods allow active components of the community to be
analyzed. Data generated from root samples are currently
the norm in molecular AM fungal community ecology. This
reflects the interest in the plant–fungus association and also
the difficulty in extracting AM fungal DNA directly from
soil (Gamper et al. 2004). The major advances brought about
by DNA-based studies increased understanding of the global
biodiversity of AM fungi (Öpik et al. 2013; Davison et al.
2015), and improved knowledge of their taxonomy (Schüßler
et al. 2001; Oehl et al. 2011; Redecker et al. 2013). These are
the prerequisites to studying community dynamics of AM
fungi.
1.2.2 From Community Fingerprinting
to Deep Sequencing
The first eukaryotic nuclear ribosomal RNA (rRNA)
gene sequences (Medlin et al. 1988) and subsequent design of
universal polymerase chain reaction (PCR) primers for fungi
MOLECULAR COMMUNITY ECOLOGY OF ARBUSCULAR MYCORRHIZAL FUNGI
(White et al. 1990) led to the first eukaryotic 18S rRNA gene
sequences from Glomeromycotan fungi (Simon et al. 1992).
The development of PCR-based techniques started molecu-
lar taxonomy of AM fungi and enabled linking phenotypic
data (mostly spore morphology) with genotypic data (DNA
sequences) and yielding better understanding about phyloge-
netic relationships of Glomeromycota. Early studies of AM
fungal community diversity were performed using cloning
and sequencing (Clapp et al. 1995; Sanders et al. 1995). The
next big development was the design of fungal primers that
exclude plant sequences (Helgason et al. 1998). The paradigm
shift driven by these studies was the unambiguous evidence
that multiple colonizations, that is, several AM fungi cocolo-
nizing a root space, even in short root lengths (Helgason et al.
1999), were widespread and that AM fungi are nonrandomly
distributed among their host plants (Helgason et al. 2002).
The shift from spore identification to study AM fungal DNA
and RNA in roots meant that active components of the fun-
gal community could be analyzed. Furthermore, cloning and
Sanger sequencing permitted detection and identification of
multiple co-occurring AM fungi in situ, without the need for
recognizable morphological features.
It is important to highlight the fact that AM fungi are
a monophyletic fungal group (Phylum Glomeromycota),
unlike fungi forming other mycorrhizal types; the design
of group-specific primers is easier. Such primers helped
identify AM fungi in planta, excluding plant sequences
and sequences of non-AM fungi colonizing roots and rhi-
zoplane. Several other primer sets specific for AM fungi
as a group or smaller subsets of them (e.g., families) have
been designed, further improving the detection of AM fun-
gal diversity (Redecker 2000; Lee et al. 2008; Krüger et al.
2009; summarized by Hart et al. 2015). Improvements in
primer systems have made the large-scale field studies pos-
sible by enabling capture of almost all of the diversity of AM
fungi in studied ecosystems.
Co-occurrence of multiple AM fungal species in a (root
or soil) sample is common. Quantifying community com-
position necessitates the separation of PCR products of
individual fungi by molecular community techniques, by
using either fingerprinting or DNA sequencing methods.
A range of fingerprinting techniques have been applied to the
study of AM fungi: polymerase chain reaction–restriction
fragment length polymorphism (PCR–RFLP; Helgason
et al. 1999; Husband et al. 2002; Vandenkoornhuyse et al.
2002); single-stranded conformation polymorphism (SSC;
Kjøller and Rosendahl 2000; Nielsen et al. 2004); terminal
(t)-RFLP (Vandenkoornhuyse et al. 2003); denaturing
gradient gel electrophoresis (DGGE; Kowalchuk et al. 2002;
Öpik et al. 2003); and temperature gradient gel electropho-
resis (TGGE; Cornejo et al. 2004). The advantage of these
techniques was rapid and relatively inexpensive profiling of
AM fungal communities; however, without sequence data,
the comparison and re-evaluation of individual studies are
usually not possible.
5
In the early molecular AM fungal community studies,
there was a trade-off between the high sample throughput
but low study-to-study comparability offered by fingerprint-
ing techniques and the costly choice of cloning and Sanger
sequencing to identify individual fungal taxa, providing
easily comparable and reanalyzable sequence data. High-
throughput sequencing (HTS) or next-generation sequenc-
ing (although this term is almost outdated as there is now a
newer generation of sequencers present, Kircher and Kelso
2010; Venkatesan and Bashir 2011) was initially costly and
had low sample throughput. This has now developed to
enable both high sample throughput and sufficient sequenc-
ing depth per sample at affordable costs to mycorrhizal
ecologists. High-throughput sequencing techniques, as they
sequence by synthesis, have also removed the need for sepa-
ration of individual PCR products either via fingerprinting or
via cloning techniques. HTS yields more accurate data about
rarer members of AM fungal communities via increased
sequence numbers per sample (sequencing depth), thus typi-
cally reporting higher richness values (Öpik et al. 2009). It is
noteworthy that typical root or soil samples used in AM fun-
gal community surveys contain an average of 5–40 species
(operational taxonomic unit [OTUs], molecular taxa; Hart
et al. 2015), which is lower than the reported richness values
of general fungal (Toju et al. 2013) or bacterial communities
(Mantar et al. 2010). Therefore, the sufficient sample-based
sequencing depth is lower in the case of AM fungi than that
in some other soil microbes (Hart et al. 2015).
The shift from cloning and Sanger sequencing to HTS
approaches has been both disruptive, completely changing
the scale and design of field-based experiments, and trans-
formative, revealing a new understanding of AM fungal
diversity and dynamics. High-throughput sequencing can
now be used to relatively rapidly profile dynamics of AM
fungal communities in large-scale field studies to describe
temporal (Dumbrell et al. 2011; Cotton et al. 2015) and spa-
tial (Öpik et al. 2013; Davison et al. 2015) variations in these
communities. One of the transformative results stemming
from HTS data has been the change in understanding about
associations between AM fungi and host plant species. Early
evidence on AM fungal-host plant species level specificity
or preference (Vandenkoornhuyse et al. 2002, 2003) may be
better explained by preference among ecological groups of
AM fungi and host plants (Öpik et al. 2009).
The swift accumulation of DNA-based AM fungal com-
munity data sets has revealed diversity patterns from local
to global scales. The first AM fungal biogeographical meta-
analyses described diversity patterns related to biome, spatial
(continents), and environmental (edaphic and climatic) fac-
tors (Öpik et al. 2006, 2010; Kivlin et al. 2011). These were
followed by HTS-based large-scale case studies, revealing
lower global endemism of AM fungi than what was thought
earlier (Öpik et al. 2013; Davison et al. 2015). Observations
made in early sequencing studies, such as the dramatic
decrease in AM fungal richness related to anthropogenic
6 THE FUNGAL COMMUNITY
land use (Helgason et al. 1998), are challenged by data from
HTS studies (Xiang et al. 2014; Vályi et al. 2015). Novel
understandings are also host plant diversity-related dynamics
of AM fungal diversity in long-term ecosystem succession
and retrogression (Martínez-García et al. 2015) and divergent
impacts of invasive plants on local AM fungal communi-
ties (Moora et al. 2011; Lekberg et al. 2013a). There is also
renewed interest in spores: it appears that sequencing soil
spores, hyphae, or root-colonizing AM fungal structures
can reveal rather different AM fungal communities (Varela-
Cervero et al. 2015). Analysis of AM fungal spores can
therefore provide useful information about life histories
of AM fungi, which is not available from sequencing of
“anonymous” root or soil samples alone.
1.2.3 From Taxonomic Expert Knowledge
to Sequence Databases
The application of molecular methods to the study of
AM fungal diversity has qualitatively changed this field
of research. Accuracy of AM fungal sequence identification
has increased, as common protocols for molecular iden-
tification have been developed (Öpik et al. 2013; Davison
et al. 2015). An increasing number of studies on AM fungal
communities do not use morphological identification at all.
However, a reconciliation of the molecular and morphologi-
cal approaches can be very beneficial for an increased under-
standing of AM fungal biodiversity and diversity patterns.
A key development has been that taxonomic identification
of environmental AM fungal (as opposed to taxonomy-oriented)
samples is now reliant on the similarity of a sequence to a pre-
viously sequenced isolate, clone, virtual taxon (VT), or opera-
tional taxonomic unit (OTU). A large number of AM fungal taxa
are now known only in the molecular form (van der Heijden
et al. 2008; Öpik et al. 2010, 2014). Public sequence databases
have become essential for accurate taxonomic identification of
newly generated sequences and for comparison across studies.
Despite this, the public sequence repositories remain poorly
curated for Glomeromycota, containing low-quality sequences
or sequences carrying false taxonomic assignment. The use of
well-curated databases such as MaarjAM for Glomeromycotan
nuclear ribosomal small subunit (SSA) RNA gene, internal
transcribed spacer (ITS), large subunit (LSA) RNA gene, and
other markers (Öpik et al. 2010) and UNITE for fungal ITS
sequences (Abarenkov et al. 2010) is the standard solution used
in fungal community ecology. In addition, public sequence
repositories are developing curated subsets of their data such as
RefSeq (NCBI) (Schoch et al. 2014).
With improved sequence identification, and standard-
ization across studies, increased objectivity is now possible
in answering research questions about AM fungal ecology.
Molecular studies have increased the rate of AM fungal
diversity data generation. The databases themselves have
become an additional source of information due to their rich
metadata about source habitats, hosts, and other information
linked to records of AM fungal sequences (Öpik et al. 2010).
Increasingly, meta-analyses are performed, using such data,
to gain new insights into AM fungal diversity or function.
Examples of this include relating host plant phylogenetic
relatedness with similarity of their associated AM fungal
communities (Veresoglou et al. 2014), culturability of AM
fungi with their habitat and host types (Ohsowski et al.
2014), and obtaining the habitat or host ranges of the fungi
detected in specific data sets, in order to interpret results of
a specific case study (Öpik et al. 2009; Moora et al. 2011;
Merckx et al. 2012).
1.2.4 Molecular Quantification of AM Fungi
Quantification of AM fungi previously relied on the
microscopic assessment of spore (Daniels and Skipper
1982; Oehl et al. 2005) and hyphal (Jakobsen et al. 1992;
Sylvia 1992) densities in soils, root colonization levels
(Giovannetti and Mosse 1980; Vierheilig et al. 2005), bio-
chemical measurements of AM fungi indicator fatty acids
(Olsson 1999), detection of ergosterol (Hart and Reader
2002), or staining of chitin (Bethlenfalvay and Ames 1987).
While many of these methods are informative about abun-
dance of total fungal community or AM fungi, the methods
lack fine-scale taxonomic resolution and therefore the abil-
ity to identify species-specific responses. Quantitative PCR
(qPCR) can provide relative quantitative assessment of AM
fungi (Gamper et al. 2008; Thonar et al. 2012). If primers
are designed for specific genes, qPCR can also be used to
measure both abundance and function (Smith and Osborn
2009). However, most qPCR studies for AM fungi have tar-
geted the rRNA genes, which can have variable copy num-
bers between taxa (100–200+ copies) (Corradi et al. 2007),
which would yield biased relative abundance estimates.
Furthermore, AM fungi may have several divergent copies
of rRNA genes within their genomes (Lloyd-MacGilp et al.
1996; Lin et al. 2014). This is further complicated, because
AM fungi have coenocytic mycelia and multinuclear spores.
For these reasons, single-copy genes may be more appropri-
ate for qPCR-based quantification of AM fungi.
When designing primers for quantification of AM fungi,
it is difficult to find conserved genomic regions that would
not target any other fungi. Such primers with imperfect
specificity would therefore overestimate abundance of the
target group (Thonar et al. 2012). Once a primer set has been
designed and tested in silico against various sequence data-
bases, the extensive marker verification, usually on cultured
organisms, is required to ensure that nontarget organisms
are not coamplified. For this reason, qPCR techniques are
not widely used in AM fungal research at the community
level, which is in contrast with the technique’s broader use
for bacteria (Smith and Osborn 2009) or whole fungal com-
munities (Prévost-Bouré et al. 2011).
The use of qPCR methods have thus far been restricted to
measuring abundance of AM fungi in simple experimental
MOLECULAR COMMUNITY ECOLOGY OF ARBUSCULAR MYCORRHIZAL FUNGI
systems, using two to four AM fungal isolates. Owing to this
focus, the designed qPCR primers tend to target only a sin-
gle species (Gamper et al. 2008, 2010; Thonar et al. 2012).
A potential problem with qPCR is interreaction variability,
and therefore, comparison between primer sets and different
reactions may be inaccurate. The PCR probes (TaqMan®)
labeled with fluorophores, used in conjunction with qPCR,
allow the detection of multiple probes simultaneously. This
technique enables up to four different primer pairs to be used
in a single PCR reaction, and thus comparison between data
generated from the different primer sets is possible (Smith
and Osborn 2009; König et al. 2010). Other markers have
been used to target single or multiple species under labora-
tory conditions such as mitochondrial genes (mt) (Krak et al.
2012) and ß-tubulin (Isayenkov et al. 2004).
Despite the apparent lack of correlation between gene
copy number and biomass (Gamper et al. 2008; Shi et al.
2012), qPCR techniques have revealed interesting results
about interactions among AM fungal species. The use of
qPCR of mtLSU taxon-specific markers has suggested
that AM fungi that provide little host benefit (“cheaters”)
can persist in the community owing to diverse mycorrhi-
zal networks and can increase in abundance as diversity
of both fungi and plants increases (Hart et al. 2013). The
use of qPCR has also provided further understanding about
competition among AM fungi, suggesting highly complex
interactions among AM fungal species, depending on the
combination and abundances of the species, resulting in dif-
ferential host plant growth (Thonar et al. 2014).
Design of a reliable and reproducible qPCR primer set
specific for AM fungi as a group would benefit molecular
quantification of these fungi in the field and in complex
experimental conditions. Such primers should be tested
in terms of how the obtained AM fungal DNA quantifica-
tion correlates with other estimates of AM fungal biomass
such as root staining and microscopy, fatty acid, or other
measurements, if available. The prospect of finding a suit-
able primer combination is improved by the increase in the
availability of AM fungal genomic sequence data. However,
the uneven distribution of nuclei in the AM fungal biomass
(Gamper et al. 2008) suggests that DNA quantification may
not be a good proxy for AM fungal biomass. On the other
hand, finding a molecular marker for AM fungal activity
(active biomass) may be a more reachable target.
1.2.5 Stable Isotope Probing for AM Research
One of the great challenges in microbial ecology is to
link taxonomy to function. Analysis of AM fungi has shown
that they have multiple functions in symbiosis with the host
plants. These can be nutritional functions (soil nutrient
uptake by AM fungi and transfer to the host plant, and use
of plant’s carbon by the AM fungi) or nonnutritional func-
tions (abiotic stress alleviation and pathogen defense). The
ability to determine the contribution of each AM fungal
7
isolate or species to a given function would allow more pre-
cise measurement of symbiotic functioning. While meth-
ods exist for quantifying movement of compounds between
symbiotic partners (nutritional functions), there are no good
approaches as of now for measuring nonnutritional func-
tioning of AM fungi.
The transfer of carbon between the host plant and the
AM fungal community has been studied (Solaiman and
Saito 1997; Bago et al. 2000; Hammer et al. 2011). Stable
isotope and radioisotope tracer techniques (Johnson et al.
2002) have been used widely in fungal research to track the
flow of carbon from host plants into the AM fungal commu-
nity (Walder et al. 2012; Fellbaum et al. 2014). These stud-
ies use isotopically labeled “heavy” carbon (13C) or nitrogen
(15N) compound substrates such as 13CO2 and trace the flow
of the labeled carbon into the biomass of the plant and then
into AM fungal biomass by using isotope ratio mass spec-
trometry (IRMS). Alternatively, radiolabeled 14CO2 can be
traced using a scintillation counter. Fine temporal scale
sampling, combined with tracer studies, reveals the rela-
tive transfer time of carbon from the host plant to the AM
community and resource allocation of photosynthetic car-
bon by the host. Tracer approaches have also been used for
15N-labeled compounds to investigate the transfer of nitro-
gen from earthworms to the AM fungi and then to the host
plant (Grabmaier et al. 2014). Nitrogen tracer studies have
been informative, but less is known about the role of nitro-
gen nutrition in the AM system and its impact on the sym-
biosis (Hodge and Storer 2015).
Radioisotope and stable-isotope tracer approaches have
been revealing about symbiotic processes between host plants
and AM fungi. There have been few attempts to measure how
the individual AM fungal species use carbon compounds
produced by a host plant, because this is methodologically
challenging. The host plant transfers carbon to the AM fungi
in the form of simple sugars such as glucose (Bago et al.
2000). The exact nature of carbon compounds utilized by the
fungus, however, is not known, as they have not been directly
linked to specific AM fungal species. It is also unknown
whether different AM fungal species utilize the same carbon
compounds or have a preference for certain sugars. In other
study systems, similar information has been gained using
stable isotope probing (SIP) (Radajewski et al. 2000). Like
tracer techniques, isotopically labeled “heavy” carbon (13C)
or nitrogen (15N) compounds are added to the system, and the
flow is tracked into specific biomarker compounds such as
phospholipid fatty acids (PLFA) (if 13C is a label) (Boschker
et al. 1998) and nucleic acids, DNA (Radajewski et al. 2000)
and RNA (Manefield et al. 2002), for 13C and 15N.
PLFA-SIP is a highly sensitive technique, because even a
small change in isotope ratio in PLFAs can be detected after
a short incubation time with a labeled substrate. The method
can therefore provide semiquantitative information about
taxon abundances. However, taxonomic resolution obtained
via PLFA-SIP is low, because fatty acid markers are specific
Another random document with
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paarse en blankhelle, blauwe gebloemten van àl wild
gewas, in ’t heet-zonnige gras. En stiller al, op den
doodstillen Zondag, heilig goud-droomden de
goudene lommerende oprijpaadjes; blakerden de
tuindershoeven, als van den weg af, naar achter
geschoven, brandend tusschen de felle zegeningen,
van zijïg groen, tusschen fluweel malsch-groen, ’t
stugge, geripste, ’t gladde, ijle, zacht-trillende, even
wuivende groen, ’t licht gepolijste, vonkende, ’t
goudlicht-afbrandende groen, ’t groen van al soorten
boomen en bladeren.

Daarin gezakt, in zwijm van zomerende glansen,

laaiden òp de lakrooie dakjes, tegen hel hemelblauw,
brandende lakgloed in ’n trillende lichtzee van azuur,
van ver wazig bedampt in hette van goud en bevend
hemelpaars, achter boomen en rijzen. [243]En vooráán,
op de laantjes, als vreugde-oogen, de
kolorietraampjes van hoeven, dichtgewolkt met ’t
blanke, innig-knussige, spul van wit gordijntjes-
neteldoek, en ’t fijn-kleurig spul van blommetjes er
onder op postrand. Hier en daar èven bezond soms, in
gouden gloei vlamden de kleine raamruitjes in de
heete praal van geraniums, hellerood, en late irissen.
Vóór verweerde groen-bemoste pompjes, sliertten
boerschige perkjes, in toch plechtige warme
kleurdiepte. En langs de lage muurtjes, in zonnekartel
en flitsbeef aangegloeid, muurtjes van verweerd
oranjebrons en pracht-roestig bruin,—of fèl-wit gekalkt
in zengblaker,—dartelde één heet vlammenspel van
indische kers, omboogd in fijn gestrengel en getak. De
oranjevlammende, de rooie, diep en vuurheet,
zengend den muur, daar opgegroeid in kleurpoortjes,
 omhuivend de lage geveltjes in cierbuig van
kruisraam, tot boven ’t ruitje, waar de takken elkaar
verstrengelden rond ’t dak; vlammige guirlandes, heet-
zonnend en daver-gloeiend tegen de roodsteenen en
hel-witte kalkmuurtjes in hellen bloemenbrand, van
zomerfleur omkranst.—

Zoo, de hoevetjes in hun jubelend dakrood, stonden er

gegroeid tusschen laaggroen, bloem en licht. En de
zomer, de heet-heerlijke, brandend-joelende zomer,
geurde en klotste, golfde en sproeide er om heen,
tegen aan, in kleuren en vlammenzon, in lucht en
lommer, in gloed en ruisch van heet leven. En àl
warmer, in ’t groen-gouden laandiep, gloeiden òp de
zijlaantjes en dwarse weggetjes, rullig bespoord en
begroefd, in de zomer-rookige zonnehette, die
knetterde en vlamketste als toovervuur in ’n
reuzenhaard, de hel bebloemde huisjes begloeiend.

In Zondagsche vroom-gouden stilte, blakerden de

geveltjes en daakjes, en sommige raampjes azurig
belommerd, keken half schaduwstil in schemergroen
uit, op de zonnige paadjes. Er achter, de tuinderijen en
boomgaarden, wijd omhegd in groen raster van
hagedoorn, brok aan brok in één gouden nevel van
stroomend licht,—brok aan brok daartusschen in koele
schaduw van verduisterend boomgroen,
sprookjesscheemrig van lichtval dat hing als ’n
wolkschijnsel, in teeren droom van uitgedoofde
glansen. En verder op, naar ’t vlakke duin, aan laan-
eind, stonden [244]kaler, ten voeten uit, wat hoeven,
tusschen rijzen, fel-bloot en aangevreten heet van
zongloei, tègen den zengend-blauwen lichthemel. Eén
 ruisch van teer rose wuifde òp uit slinger-laantje van
duizendschoon-boeketten.—Wat verder weer, heet-
gebrand, kalk-blanke geveltjes aanéén,
samengeschroeid in den zonnenden blink van licht,
geel-okerige muurtjes, omstrooid, volgeworpen van
boven tot onder, met goudgele, diep oranje-roode en
bleek-purperen indische kers en goudsbloem.—Op
zon-fel erfbrokje, dat achteruit openlag te zwijmen van
hette, kronkelde wild geslinger van vlammezonnetjes,
goudsbloementuil, wild en woest, heet tusschen
grasgroen. Dáár plots, drie huisjes bijéén, in brand
van goudsbloem en kers. En tusschen de wond’re
ronde bedauwde blaadjes, het vlammige brio, hoog
langs de voorgeveltjes, in oranjerie-gloed, afkaatsend
licht op vermorzeld, bemost rommelschuurtje er
tegenover.

En heel eenzaam, bij ’n laag-glooienden duinhoek,

zonde op, één geslonken verweerd hoevetje, ’s
winters bang gebombardeerd door storm en zomers
verzengd, vervreten van zon, nù, om en òm bestrooid
met zomerzonnige flox, paars-gloeiende en zoet-
roode duizend-schoon; kers en goudsbloem, slingers
van blanke windekelken, beschuimd van licht, wonder-
stil omruischt van zondagsvroom.—En verderòp, aan
weerszij, de tuinen, de tuinen met trosgloei van
wonder-besjes, overal, overal, hel-rood, glazurend-
rood, glanzig befonkeld en bezond, tusschen gouden
schemerend lommergroen. En zacht onder ’t
struikgewas, de aangroei van frambozen, dof purper,
donzig en bleek nog. Zóó in eindeloosheid van stilte,
blakerde de zondagochtend, achterland van ’t
 stedeke, met z’n gaarden en vruchten, in purperig
vergloeiend toovervuur.

Onverschillig de Hassels liepen door, vreemd zich

voelend in hun stil gekraak van beschoende voeten,
gewend aan klompklos en luwte van kousen. In ’n
zwaai van den weg, belandden ze aan de haven en
Dirk, met ouë Gerrit en Kees achterna, koerste al op ’n
kroeg áán. Vol zat de herberg van biljarters en
drinkers. Scherpe jeneverstank zuurde in ’t lokaal en
roezemoezig de opgemonterde tuinders en knechten,
stemmeraasden [245]om ’t biljard. Beschonken kerels,
in zeurd’rige klets, belamenteerden elkaar, en vroeg in
Zondagsjool stormde ’t landvolk in. Met
deftigheidsallure, de hoeden schuin op gekapten kop,
bewogen ze zich onhebbelijk en harkig in d’r deftige
kleeren. Ringspul glinsterde op handen en kettingen
schommelden op vesten. Reuk van versch linnen
geurde òp en van allen kant blankte overhemd-wit. ’n
Paar gemodernizeerden met rooie dassen, hoog-
geboord, werkten zich harkig de handen uit knel van
manchetten. Er was gedol en afgunstig geblaas tegen
de chiekdoende klungels, en geschreeuw dat ze maar
naar ’t stations-koffiehuis bij Termeer mosten biljarten
en niet hier, onder hullie schorremorrie.—

—Jai en Joap, jullie hoort d’r puur bai de deftighait!

debies! je laikt d’r ’n domenie.. wai hebbe veur jou
gain mond fraite hee?

Dirk was dadelijk bij aankomst naar de toonbank

geloopen, bestelde voor hèm en den Ouë klare met
suiker. Schuw keken de kerels bij Kees’ áánstap. Daar
 ha’ je f’rduufeld de Strooper. Maar nou geen angst,
want ’t was klaarlichte dag, en Kees werkte al den
heelen zomer fesoenlik, gelijk met hen op, in ’t land.
De spokige lucht was van ’m afgewaaid.

Gewoontjes zagen ze’m alles doen als zij zelf en niks

niemendal geen geheimigheidjes meer. Voor z’n
strakken kop angstten ze nog beteuterd en voor z’n
zwijg, z’n moker-vuisten en z’n reuzig lijf. Maar toch, ’t
was d’r puur geen kwoaje kerel, als je’m maar in z’n
waarde liet.—

Naast Dirk waggelde ’n groote kerel, stronkige

haaibaai, met bezopen tronie en lodderigen staar. Hij
bedelde om ’n borrel, in strompelenden
woordenstotter.

—Jai hep d’r g’nog, jai kakketoe! zei norsch-ernstig

kastelein, ik tap jou nie meer.… je ken nie betoàle.…
en jai ken d’r nie meer hebbe, ook nie! ’t is d’r f’rdomd
pas half tien en je ken d’r nou al je neus nie meer
finde.… debies!

Als ’n drenzerig kind, huilerig en pruilend, zeurde de

kerel door, opbonzend met z’n herkules-lijf tegen den
kleinen kastelein, die telkens zacht achteruit
schuifelde. Smeeken [246]deed ie om ’n borrel,
smeeken z’n stèm, z’n beverige begèer-handen. Maar
norsch-streng bleef kastelein weigeren.

Van den een, schoof de reus naar den ander,

bedelend om ’n vrij slokje, pruilend huilerig en smart-
zwaar geslagen van melancholie, bewerend dat ie op
lange na nog niet dronken was. Maar iedereen stootte
 ’m op zij. In dronken drift en wanhoop waggelde ie
eindelijk naar de deur, vuistdreigend den kastelein, die
in hoonlach schoot, schoueropschokkend met
minachting.—

—Se.… moste.… mo.… ste.… jouwes.… n-n-n.…

spòa.… tussche.… de rib.… be.… stootte.… jou.…
jouw geep!.… stotterde drinkeboer, en rinkelfel
dreunde ie de deur achter zich dicht in smak.

Kees wou niet meer dan één borrel. Ouë Gerrit dronk
zoetjes, in vadsige lekkerheid. Maar Dirk alleen
genoot, roerde z’n brandewijntje met suiker, een na
een, kneep half dicht z’n oogen van pure lekkerigheid.
Vijf na elkaar had ie ’r ingeslagen, dat z’n lobbeskijk al
zachtjes-an in guitig spel van licht verfonkelen ging.
Hij lolde met de kerels aan ’t biljard, zei hoe ze stooten
nemen moesten en waar ze heenrobbelen zouden.—

—Kaik.… kwankwinker! ’n deurskieter, van bòve roàke

mit ’n luisje effect.. kaik de balle stoan,.. achter
malkoar!.. krek de oarepels in ’t duin.. Nou stoot toe!..
’t is je suster nie! hai mot d’r boofe òp se pèt!—

Kees wou weg, sleurde met duwen Dirk mee. Bij de

deur botsten ze tegen den grooten dronken kerel òp,
die met verzopen huil-lach-gezicht en grauwen kop,
als uit stopverf gekneed, strompelend kwam
inwaggelen.—

Hij gierde, kwijlde van pret en juichend hield ie ’n

dubbeltje tusschen z’n smerige dikke vingers gekneld.
In zwaai-waggel sliertte hij op den kastelein àf, achter
’t buffet, en dreigend met schorren uitdaagklank in z’n
stem, raasde ie:

—Nou.. is.. is ’t tug-glad-en-al mis!.. hei maa’n! Nou..

he’k.. he’k veur ’n poaretje hee?

—Nou! je bent main d’r ook ’n feestnummer!.. gierde

de kastelein, die nog niet schenken wou.

—Wa.. wá’ nou!.. mì’ de lood.. loodpot.… nie-nie..

[247]fe.. soene.. soenelik.… in je.… i.… in.… in je
klauwe!.. da.… d’á hep je tug.… tuu.… u.. ug glad-en-
al.… àl-àl.… mis!

—Seker, moar je stoan d’r nog in ’t krait.… jai

kabbeloebeloap.… goan d’r bai ’n aer!… ik sien
lieferst je hiele oa’s je toone.… mó’ je d’r nou al de
blommetjes buite sette seldere-mosterd!

—Tu.… tu.… rogge.… ge.. ge.. rogge.… megoggel!

hou d’r je fesoen he.… ikke.… bin.. d’r ’n nette.… vol..
vòlbloed.… nette.… nette maa’n.… enn.… enne … jai..
jai.. kaik! hier.… hier.… is main dubbeltje.… noù?.. wa
sàit.… sai it tie noù?

Wat lachende kerels bemoeiden zich in ’t geharrewar,

hadden lol in ’t dronken verweer, ’t gestamel van den
lossen werker.

—Jai hep d’r al vroegies de prins sproke maa’n!

—Nou.. waa’ a ’t? ikke.. ik.. ke bin.. bin d’r ook feur..
feu.. ffeur.. ffeur.. den donder.. feur! feu.. feur de
koning Fillim Drie! Laife.… lai.. fe Fillum dr.. drie.…
daa’s.. daa’s main weut!.… hee?.… enne.. bi.… bi.…
 jai.… d’r.… nooit.… nie.… nie iet.… in.… in de wind
weust.… hee?

Kees stapte ’t eerst uit. Hij had ’t benauwd; hij kon die
kroeglucht niet langer inademen, omdat ie ’r nooit
kwam. Ouë Gerrit was lichtelijk draaierig van z’n
dronkje, voelde zich blij weer op straat te staan. Alleen
Dirk was weer teruggestrompeld, bleef plakken bij de
biljarters.

—Nee Ouë.… jai stapt d’r noar domenie hee?.. bai

tiene; moar.… ikke lief dá’ nie.. ikke sit d’r lieferst
hier.… in de vaif sints-kerk.… wa’ jou? hoonde Dirk
den Ouë na, die achter de deur hem nog wou
meetronen.—

[Inhoud]

II.

Er was weer overstrooming van werk bij Ouë Gerrit,

en Kees had ’t bij Dirk gedaan gekregen, dat Ant mee
plukken mocht. [248]Eerst had z’n vrouw gevloekt en
geraasd, dat ze bij die ketters, die vuiliken, geen stap
doen zou. Maar met d’r zwanger lijf wou d’r niemand
meer hebben in de haal. En ’t moest, moèst voor wat
duiten, nou Wimpie al zwakker, meer versterkende
middelen noodig had, veel meer dan anders. Ouë
Gerrit had gegromd, voelde met dat roomsche tuig als
zwaarder kruis en bijgeloof-ongeluk op z’n dak. Guurt,
 al verafschuwde zij Ant, kon ’t niets schelen en de
jongens keken nauw naar schoonzus òm. Zoo kwam ’r
wat meer verdiensten bij Kees. Toch zat Ant, met haat
en wrok in ’r lijf, dichtgebeten mond van spraakloozen
nijd, bessen en aardbeien te plukken. Dientje en
Jansje d’r twee meisjes, die ze anders nooit ’n stap liet
zetten, in huis bij Kees’ vaar, mochten in den ochtend
helpen voor ’n prikje bij den Ouë, omdat ie hield van
klein goed, dat niets vroeg en véél werkte, vooral
meisjes. ’s Middags hurkten ze bij ’n ander tuinder in
Duinkijk, voor ’t wieden van dahlia’s, heel jong nog, en
voor de pluk.—

’t Weer was omgeslagen op ’t laatst van Juli. Veel

regenbuien in zwaren ruisch neerstroomend, kletter-
kakelden tusschen de bladeren en boomen; grauwe
onrustige lage luchten verduisterden droef ’t
Wierelandsch groen. Met schrik en beven loerde Ouë
Gerrit naar z’n groote lap boonen en spersies. Daarin
zat z’n bestaan voor dit jaar. Hij had vier millioen stuks
te leveren aan de fabriek en ’n geweldige hoeveelheid
noodig voor eigen verkoop nog op de groote stad. De
vruchten, appelen, peertjes, bessen en frambozen
konden ’m eigenlijk geen zier schelen. Dat was
kleingoed. Maar nou, met den omdraai van ’t weer,
vloekte ouë Gerrit omdat ie de vrucht van z’n boonen
niet zag zetten en heel veel bloesem al verrotten.—
Dag op dag bleef gure winderigheid snerpen, en
gietbuien die eerst als drenkende lafenis op ’t heet-
uitgedroogde land neergeruischt waren, hielden áán,
angstiglijk en lang, dat den tuinders de schrik om ’t
hart sloeg. Soms, tegen den avond kwam de zon even
koekeloeren achter grauw gewolk, bleekte weer
 dampig weg, in nattig grijs. Bang verklonken vage
stemmen over den oogst.—En Ouë Gerrit leek ’t
bangst van allen, brandde zich aan z’n benauwing,
[249]die wurgender òpklom naar z’n strot.—Plots tegen
eind Juli keerde ’t weer. Zon kwam luchten-nevel en
zilver-dampig regengewolk doorgloeien, doorpoken
met licht. En harder weer gromde de zwoeg rond.—

Ant, met ’r zwaar-zwanger lijf kroop tusschen de

bedjes, zuchtte en kermde gesmoord, keek stroef en
wrang-nijdig als Guurt of Piet d’r wat vroegen van de
pluk. Met gift en haat in ’r, werkte ze bij dat tuig, dat
nooit naar d’r Wimpie vroeg, nooit nog wat voor ’t
schaap hadden meegegeven en voor de kinders,
Dientje en Jansie, geen bakkie koffie overhadden.

In woede en stomheid kroop ze, akkerbed na

akkerbed af.—

Tegen acht uur op den avond, vergoudde de stille

aardbeihoek in paradijselijken glans. Fijne geurige
zomerdampen nevelden òp, van den eindeloozen
akkerkring rondom, floersig en vochtig van glansen
met ’t onderbroken zicht op de verte van àl groen
gehaag en zonnige elzensingeltjes. Zonnedaal
verjongleerde z’n roode toortsen, tooverig vuur en
goudrag tusschen ’t avondgroen, en gloed bloedde
over den hurk van plukkers en pluksters, verspreid
rond de bedden.—

Ouë Gerrit joeg òp omdat ze hèm joegen. Kees werkte

op ’n doodstil punt, tegen ’n weibrok áán met Dirk,
tusschen de kruisbessen. Achter hem stonden wat
bedden uitgeschoten aspersie in brand, ’n goud-roode
 donzige wonderwolk, waar ’t heilige zacht-vurige licht
doorheen webde ragste glanzen. De lage wolk gloeide
al rooder, dwars door de schaduw-lengte der
bewegende kerels, die er omheen sjouwden met
verduisterende gebaren.

Gehurkt voor de doornige kruisbessenstruiken, ristte

Kees de goudgele vruchtjes naar zich toe.—’n Klein
mager, stakkerig meisje van tien hielp plukken en de
mandjes opkoppen,—

—Hoeveul stoan d’r in de bak? vroeg Kees, diep-

stemmig in de avondstilte.

—Tien.… twintig.… dertig.… acht-en dertig mandjes,

telde ’t kindje.

—Nog vaiftien, dan is ’t daan,.… goan jai d’r nou moar

[250]an de besse.. pluk d’r mooie.… veertig mandjes.…
of wacht! help d’r nog rais twee hier.

’t Kind, goudkrullig kopje, met smoezige wangetjes,

morsig als van ’n vuil elastiek popje, luisterde naar
Dirk, stond in gewilligen stand klaar, om daadlijk te
doen wat ’r gezegd werd.—Van bessenhoek kwam
Piet aanstappen.

—Bin je ’r t’met hier?.. ké’ jai de klaine misse?.…

f’rdomd aa’s je d’r nie vast veul ferder mee komp aa’s
mit ’n knoap! Die loâje je t’met ieder keer in de staik!
Da là d’r aige omkoope.… feur ’n kwartje.… ’n
dubbeltje per dag meer.
—En gain drommel dá’ hullie beskiete.… wrokte Dirk
mee,—aa’s luie meroakels in de son.. die loope d’r
puur de heule dag op de kantjes.… Nou.. moar de
maid ken d’r Kees nog nie misse.… so mot t’r nog
veertig besse-mande plukke t’met.

—Aauuw! pijnschreeuwde ’t kindje plots, ’r hand in

den mond versabbelend.

—Wà’ nou? hai je je aige weer prikt? paa’s d’r dan òp!
jai ken vast gain bloed misse.… zei Kees norsch.

Goud krullekopje bleef met pijnverwrongen gezichtje ’r

vingers bezuigen, terwijl Kees, tegenover haar, aan
anderen struik, in ritsige scheuren de goudene
vruchtjes met kromhakende toppen naar zich toe trok.
Z’n groote handen woelden tusschen de dicht
saamgegroeide naaldscherpe doornen, of ie geen pijn
voèlde. Soms angelden vijf doorns te gelijk z’n knuist
en pols in, dat ’t bloed langs z’n handen sijpelde. Maar
dóór werkte ie, zonder klacht, zonder z’n grijpklauw
zelfs af te vegen. ’t Meisje huilde van pijn. Van drie
kanten tusschen ’r vingertjes, had ze wonden in ’r
handje gescheurd, en telkens wou ze angstig voor ’t
rood, ’r bloed stelpen en inzuigen. Heviger brandden
de vleeschscheurtjes en bangelijk ritste ze’r nieuwe
kruisbessen af, schurend weer langs de doornhaken,
bevend dat ze de wondjes verder zou inscheuren.—

—Nou maid! nou moar àn de oalbesse, dan nep je

gain stekels hee!.. moar denk ’r an … nie van de loàge
f’rdieping, die binne d’r nou fuilsandig van de raige die
teuge de grond spat hep! [251]
 Helpstertje was uit ’r hurk òpgestaan. Haar gezichtje
krampte nòg smartelijk van pijn en ’r bloedende
vingertjes beplukte en zoog ze af, onder ’t luisteren.—

Langs de struikbosjes kruisbessen die flonkergoud

gloeiden in avondschijn, prachtig en vochtig als
lichtbolletjes, overal tusschen ’t groen, wrong ze d’r
mager karkasje, schoof ze, telkens ’r vastgehaakt
rokje lospeuterend, in ongeduld vóórt, naar de rooie
bessen, die zwaar betrost glansden in glazuur lachje
van prachtrood.—

Den bak sjouwde ’r Kees na, volgeplukt met slanke

mandjes kruisbessen en ertusschen ’n nieuw soort
groen-paarsige, als groote glazen stuiters, spiralig
bekleurd van binnen.—En de goudfelle zonnige
knikkertjes er naast, in kleinere slofjes.—

Zachte zomeravonddamp waasde over de tuinderij.

Elke hoek goudde òp in rood wazigen zonneglans. De
omgewerkte grond, vol gras, onkruid en gewas,
verschemerde zacht toovervuur onder de
vruchtboompjes en rond de lage kronkel-stille
stammetjes vervloeide ’n lichtend rag, aureolenglans.
—

Telkens uit anderen hoek in den stikvol beplanten tuin

dook ’n werker òp, goud-rood overgloeid,
gereedschappen in de bronzen wroethanden gekneld.
Ouë Gerrit scharrelde met spa en hark onder de
pruim- en appelboompjes. Hij kroop half onder ’t lage
getak door, dat àl bukkend, ’n stuk van z’n blauwen
kiel in den rooden avondbrand vervlamde, dan weer
z’n baardzilver overgoot in wondren glans. De
roodgegloeide struiken en vruchtstammetjes onder z’n
voeten sidderden in den heiligen val van avondgloed.

Overal voelde de Ouë zich vastgehaakt, tusschen

brandend, getwijg en takkronkels. Maar overal wou ie
bijzijn in ’t late uur, om te weten of er wel gewerkt wier.
Aan de Beek was ie al voor ’n uur geweest en daar
had ie ’n vent weggestuurd, die ’n kwartier na het
schoft nog had staan smoken.—Nijdiger nog werkte ie
zich tusschen de struiken en vruchttakken door, en
telkens sterker gloeide áán z’n zilveren kop en
rompblauw in den rooden gloed van zonnedaal, die
daar wonderen bleef [252]onder ’t laag takgekronkel,
tusschen fijne stammetjes en avond-beglansd gras.—

—Hee, hoho! sain jullie doar bai de bèsse?

schreeuwde ie de landstilte in, hij zelf nog gekneld
tusschen de enge paadjes van gestruik en boompjes,
een hand vast aan rood-begloeide stronk. Geen
antwoord klonk terug. Harder schreeuwde ie, z’n lijf
meer opgeheven.

—Joa! klonk zangerig en wijd uit de verte Kees’ stem,

vol en diep.—

Klein Dientje, Kees dochtertje, zat aan de bessen, met

’t goud bekrulde huilebalkje naast ’r geknield, toen ’r
grootvader voorbijklompte.

—Hoho! Bai de korrels afknaipe maide en pèrs jullie

nie!

Het kind, gehurkt in goudgloei van avondschijn, vóór

de zacht-betoorste boompjes, keek òp, half geschrikt
van ouë Gerrit’s stem, omdat grootvader d’r bijna nooit
zelf aansprak.

—Je sussie.… is d’r an de oarebaie!.. en aa’s je

murrege bai de fint van Duinwaik mot, hoef je hier nie
meèr te komme.. eén boas.… of gain boas!.…
hoho!.… en aa’s je kloar bent.… viere en vaife en nie
genog.… dan vroag je oome Dirk.… of d’r feur f’oàfud
nog wâ sain mot.. huhu! nie an je foader! denk k’r an!
aêrs kè’ je wel ophoepele.… veur joù.… tien aere!.…
hoho! En murrege mi de son pirsint! en dan doalik an
de swàrte besse.… de swarte.. dè swàrte, f’rstoan?

Kindje knikte, angstiglijk òpstarend d’r kopje. De

blauwe oogjes goudlichtend, bleven kijken naar
grootvaders mooien zilverbaard; ze hoorde z’n
stemmebrom maar half en d’r naarstige handjes
plukten dóór, de glanzende bessentrosjes in ’r mandje
volschietend, vlug en plezierig,—

Kees reuzigde voorbij op ’n nauw graspaadje bij

greppel, vlak langs jonge koolen. Weer versjouwde ie
drie bakken met kruisen rooie bessen naar den
dorsch. Z’n klompen klotsten zacht, dwars door de
aarbeibedden. Aan den achteringang zat z’n vrouw
nog te plukken, met drie meisjes en Piet, ieder aan ’n
regel, krommig-ingebukt of gehurkt, tusschen de
nauwe paadjes. [253]Avondlucht azuurde in
prachtblauw kuisch en doodstil, schoon-geveegd van
wolkjes, in glansenvloed.—Zon straalde lager, in al
wonder tooverrood op den stillen aardbeihoek, op de
hurkende kindertjes en werkers. De groenende bedjes
vlamden zacht, en de rooie vruchtjes glansden en
geurden zoetelijk bedwelmend. En over de kruipende
werkers weefde en fonkelde uit, één rag, goud-
glanzend web, gespannen van akkerhoek tot
akkerhoek, omlichtend handen, gezichten en kleeren,
in tooverzachte vurigheid.—

Ant kòn niet meer. Haar zwangere zwelbuik hamerde

en trilde van leven. In de laatste maand, nog twintig
dagen, en ’t kind wàs ’r. Haar rug brandde van heete
pijn. Heel stil was ze naar ’t eind van haar
aardbeiregel gekropen, vlak bij ’n brok haag, waar ’n
appelboom laag-takkig kromde. Daar moest ze heen,
om zich op de been te zetten. Want schamen deed ze
zich voor de jolige nestjes van meisjes om hulp te
roepen, te vragen om steun, of haar op te trekken van
den grond.—’t Was ’n lange zware dag arbeid
geweest, veel te lang, voelde ze zelf heel goed, en ze
verlangde dòl naar Wimpie, hevig en gejaagd. Ze had
’m gezeid, dat de drukte van den haal niet zoo heel
lang meer zou duren, en dat ze met vader samen
thuis zou zijn. Hij had zwakjes in z’n handen geklapt,
gejuicht en met z’n zandzakken tegen ’t achterend van
z’n ledikantje gestommeld, zoo koortsig vroolijk voelde
ie zich in z’n beenen.—En als ze’n half uurtje vroeger
thuis kwam, juichte ie sterker, overrompelde hij ’r met
zoenen.—

Nou wou ze òpstaan, maar ze kòn niet. Op ’t eind van

’t aardbeibed, greep ze in stumperige onbeholpenheid
naar ’t krom-gegroeide dikke appelstammetje,
probeerde zich zuchtend en hijgend op d’r beenen te
hijschen. Maar ’t ging niet. Even van den grond
opgesjort, voelde ze haar armen slap en kramp-
 trekkerig in den pols. En met ’n smak, in klammen
uitgloei van zweet op ’r hoofd, viel ze weer terug op de
aarde, tusschen de bedjes, duizelig en aêmechtig.
Kermend was ze ingezakt, dat de kinders àchter en
vóór d’r kruipend omkeken, opschrikten, en dwars
over de bedjes naar Ant toeschoten. [254]

—Is d’r ’n ongeluk, vrouw Hassel? vroeg één bezorgd.

—Se’l d’r man hoale.. riep ’n ander ontsteld!

—Is boas Piet ’r nie? o nee.… die is d’r bai de

kruisbesse.…

—Niks gedoan!.. maissies ikke bin.. d’r.. tug soo el..

lèndig moe.… hee? hijgde Ant’s stem uit ’t gras,.…
kaik!.. wees jullie d’r ais.. tuikige koo.… ters.… en
nne.. trek.. vrouw Hassel.… erais op bain,.. hee?.. dâ..
se t’met stoan!

De drie helpertjes met hun roodbevlekte

aardbeihandjes sprongen ieder aan ’n kant, en
heschen met sterken span van lijfjes, in hijg en blaas,
zwaarlijvige Ant weer overeind, die zacht zuchtte en
van ’r voorhoofd al meer benauwingszweet afgutste.
Eerst nog waggelde ze, voelde ze’n verdoovende
matheid en prikkelende pijn razen in d’r verstramde
beenen en gloeifel kneushitte priemen in d’r stuit. Haar
knieën knakkerden en ’r kuiten beefden.—

Van af drie uur in den gloeimiddag had ze aan één

stuk geplukt, gehurkt gezeten. Nou voelde ze haar
beenen en dijen aan heete brei gebrand, verkneusd,
tegen haar rokken opschuren.—De helpertjes keken ’r
 áán, met angstoogjes en meelijmondjes. Ze zagen
Ant’s gezicht verzuchten in één smoor van pijn; en de
buik geweldig in zwel vóóruit, ademde en leefde zwaar
als ’n apart wezen, aan ’r vastgekneld.—Nou most ze
nog wel ’n uur op stap. Ja ze wist wel, dat ze te veel
gewerkt had,.. maar ’t most, mòst voor d’r Wimpie.
Waar anders ’t versterkende voedsel van daan, en
hoe konden ze wat krijgen voor den werkeloozen
winter, als noù, nou niet wat van ’t achterstallige
betaald wier?—Nou weer ’n uur op stap, naar huis.
Kees had ze zien voorbijdraven. Hij had ’r
toegeschreeuwd, dat ie nog naar de Haven most
lossen en oplaaien, dat ’t wel elf uur zou worden eer ie
klaar was.

Dientje en Jansie mosten nog helpen in den dorsch,

de mandjes aangeven.

Moederziel alleen, half dood, stapte Ant den

avondweg òp, langs ’t stille uitgloeiende duin, naar d’r
huis, waar Wimpie, op háár en Kees smachtend-stom
wachtte, wachtte en vroom [255]staarde op z’n
Jezusbeeldje en palmtakje, van z’n bedplankje
afgehaald en vóór ’m gezet; verbangd tusschen de
halfblinde, loerige vrouw Rams, en den stommen,
pruimpjes uitspuitenden grootvader.—

[Inhoud]

III.
 Volgenden dag scheen de zon weer fèl. Ouë Gerrit
was aan ’t frambozen plukken, met Piet en twee
helpertjes. Tusschen de nauwe struiken hurkte de
Ouë, stram en ingebukt vóór de frambozen, één voor
één de vruchten in de mandjes stapelend.—Boven z’n
zilverbaard, die in kronkelig zijïge karteltjes, tusschen
takkengewirwar òpschemerde, bloeiden de
frambozen, rozepurperend, bedauwd met ’n adem van
dof paarsrood, vochtig en donzig. Achter Ouë’s hielen,
rijden mandjes in zonnevlam, wonderteer doorglansd
van licht. Zoeter dan aardbei geurden ze rond, als
rozendroom van sprookjes-prinsesje. Rondom, in de
lucht vervloeide ’n teere geur van rozenzoet, wáár de
mandjes stonden.

—Heé Ouë, je ben d’r in màin regel, nou soek jai d’r
de mooie uit, en ikke hou ’t kriel!

—Hoho!.… da skaint t’met soo!.… d’r binne.…

—Nou.. set jai d’r op haide ook rais de blommetjes

buite!.. moar ikke seg ie.… daa’t soekwerrekie is.. da’
rooit na’ niks? d’r binne nie-en-veul.…

—Nou Piet aa’s jai d’r da rais an de swarte besse

gong,.. ke’k vast ’n mooie prais moake! hoho! ikke ken
d’r laifere soo veul ’k wil.. eenmoal andermoal.. aa’s
d’r nie g’nog binne.. koop ’k vàst bai op de hoàfe
f’noàfed.…

—Daa’s fain werk Ouë, jai f’rkoopt hullie an de

oàpetaik hee?.. en sullie stooke hullie hee? of hep je
hullie weer feur de ferbriek!
—Nou dà’ sit nog.… ikke hep se pattekelier!.. en.. en
feur de f’briek òòk.… hoho!.… kaik? tug mooie woar
hee? [256]pronkte Ouë Gerrit ’n mandje pracht-
frambozen in ’t zonlicht heffend, dat ’r roodpaars
dauwlicht overheen huiverde in vloeiende glansen.—

—’t Is d’r fain werk! t’met al de vurige, de vuile loà’je

sitte, hoonde Piet weer, die ’t niet verkroppen kon dat
de Ouë in zijn regel zat te plukken.—

—Daa’s glad-en-al mis maa’n! De vuile hep ikke hier

ampart! huhu! tug ’n f’rvloekt slecht joar! alles is d’r te
loat! t’mèt hep je de kerremis’ en kaik!.. die boone
rais? se komme paa’s kaike! hoho! en nerriges nog ’n
skepseltje an van fesoen.. da je d’r plukke kèn!

—Brom jai teuge sint Jan! blai da’ tie d’r weust is! de
sjèlotjes binne d’r krek uit! Ook nie te bestig! Is
aldegoar ’n rooi noa niks!… en de rooie kool f’rvloekt!
En nou.. hai je d’r nog spruitkool loate sette tusschen
vaif regels boone! Sel main d’r ’n stelletje worre.—

Piet kregel, liep dwars door ’n hoek leeggeplukte

zoete fransjes, naar de zwarte bessen. Kees stond er
al te plukken. Zwart-blauwig glansden de vruchtjes als
kraaienvoedsel, struikerig en verward, benauwenden
stank uitdampend.—

—Ikkom je d’r ’n pootje hellepe hee? Dirk is in de loate

doppers,.… wortele.… binne tog t’met daan!

—Soo? docht wel, daa’t ie nog nuwe portie soait had!